Lyophilization process and a teverelix-tfa lyophilizate obtained thereby

ABSTRACT

The present invention relates to a lyophilization process for preparing a teverelix-TFA lyophilizate, said process comprises the following steps: a) providing a lyophilization suspension by mixing teverelix and trifluoroacetate at a molar ratio sufficient for providing a microcrystalline teverelix-TFA suspension without formation of a gel, and b) lyophilizating the lyophilization suspension, thereby providing a teverelix-TFA lyophilizate. The process provides the possibility of obtaining a “pure” teverelix-TFA lyophilizate i.e. without any undesirable residues in the composition.

This application is a continuation of U.S. patent application Ser. No.17/255,372, filed Dec. 22, 2020, which is a 371 filing of InternationalPatent Application PCT/EP2019/067733 filed Jul. 2, 2019, which claimsthe benefit of European patent application no. 18181960.8 filed Jul. 5,2018.

TECHNICAL FIELD

The invention relates to a lyophilization process, to a teverelix-TFAlyophilizate obtained by said process, and to a method of reconstitutingsaid teverelix-TFA lyophilizate.

BACKGROUND

Teverelix is a synthetic gonadotropin-releasing hormone antagonists(GnRH antagonists) that compete with the endogenous neurohormone GnRH(otherwise known as luteinizing hormone releasing hormone, LHRH) forbinding to its receptors in the anterior pituitary gland. By decreasingor blocking GnRH action, the GnRH antagonist suppress release from theanterior pituitary gland of follicle stimulating hormone (FSH) andluteinizing hormone (LH).

Both FSH and LH are involved in normal reproductive function.

In females, FSH stimulates the growth of immature Graafian follicles tomaturation, whereas changes in LH levels control ovulation. In males, onthe other hand, FSH plays an important role in spermatogenesis and LHstimulates production of testosterone in the testes.

Accordingly, teverelix is suitable for treatment of hormone-dependentconditions such as benign prostatic hypertrophy, hormone-dependentprostate cancer, endometriosis and uterine myomas.

Sustained release formulations usually require very high concentrationsof active ingredient dissolved in small volumes water or some othersuitable solvent(s). Since teverelix(Ac-D-Nal-D-pC1Phe-D-Pal-Ser-Tyr-D-Hci-Leu-Lys(iPr)-Pro-D-Ala-NH₂) is ahydrophobic peptide it is not freely soluble in water or in othersolvents. Teverelix further has a propensity to form gels in aqueous orother solvents even at low concentrations, which greatly limit its usein sustained release formulations.

WO2003/022243 aims at solving this problem and discloses that theformation of a gel may be prevented by contacting the teverelix peptidewith a counter-ion, e.g. trifluoroacetate (TFA). According toWO2003/022243, a ratio of teverelix to the counter-ion trifluoroacetateof at least 1:1.6 is essential in order to ensure the desiredmicrocrystalline suspension is obtained, otherwise a gel will be formed.However, the inventors of the present invention have found that themolar ratios disclosed in WO 2003/022243 will result in both undesirablegel-formation and in suspensions which are not homogenous. This is aproblem, not only because such suspensions will be difficult to inject,but also because the bioavailability of the teverelix peptide iscompromised since the gel interferes with the desired sustained actionof said peptide.

The inventors of the present invention have furthermore discovered thatthere are batch variations in the molar ratio of teverelix to thecounter-ion TFA provided by the manufactures of WO2003/022243, eventhough the applied manufacturing conditions and processes are identicalfor said batches. Since the molar ratio is essential for obtaining thedesired microcrystalline suspension, variations in said ratio may affectthe bioavailablity of teverelix in the pharmaceutical formulations. Itis therefore essential that medical personal and other users can rely onthe molar ratio of teverelix to trifluoroacetate provided by themanufactures.

Another problem with the teverelix compositions of WO2003/022243 is thatsaid compositions are not stable during storage e.g. at refrigerationand room temperatures, and accordingly teverelix has a relatively shortshelf life under such conditions.

Lyophilization (freeze drying) is a widely used process for improvingthe stability of pharmaceuticals, and even thought WO2003/022243 referto freeze-drying and lyophilization of teverelix, the document onlyrefers to the technique in a general manner such as “the material isfreeze-dried over night” and other general statements of similardescription, but fails to disclose the specific requirements needed forlyophilization of teverelix-TFA.

Teverelix-TFA has a very low solubility in water and in order to providea solution that can undergo a lyophilization process the teverelix-TFAhas to be dissolved. However, this can only be accomplished in a strongacid, e.g. a concentrated acid. The use of strong acid as a solvent notonly requires more attention to the freeze drying process, but lowertemperatures are also required to freeze and condense the solvents andsaid solvents can easily bypass the condenser and end up causing damageto e.g. the vacuum pump. Thus, specialized lyophilization equipments arerequired in order to prevent damage to the equipment, adding to theoverall costs of the lyophilization process.

Furthermore, before lyophilization is initiated it is very important tocarefully consider all non-volatile compounds of the sample as thesewill concentrate along with the relevant active ingredient, i.e.teverelix. Non-volatile acids or bases can cause extreme pH, and thepresence of salts can result in very high ionic strength when the sampleis resolubilised. It is therefore essential that special care is takenwhen lyophilising a teverelix composition as the resultant lyophilizatemay end up comprising residual acid which will have an adverse effect onthe intended use. This adds further complexity to the lyophilizationprocess as it is important that all solvent is removed in theophilizate, in order to ensure that the reconstituted teverelix-TFAlyophilizate can be administrated safely to a patient, e.g. bysubcutaneous and/or intramuscular injection.

Finally, the viscosity of the teverelix-TFA solution causes thesterilization filters to clog also making the sterilization processlaboriously and expensive.

Accordingly, there is a demand to develop a new process of manufacturinga stable and sterile teverelix-TFA lyophilizate having a long shelflife.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1G show molar ratio results in accordance with one or moreembodiments of the present invention.

FIGS. 2 and 3 depict the results of Table 6 in accordance with one ormore embodiments of the present invention.

FIG. 4 depicts the results of Table 8 in accordance with one or moreembodiments of the present invention.

FIG. 5 depicts mean plasma levels in a four week in accordance with oneor more embodiments of the present invention.

FIGS. 6 and 7 depict stability results in accordance with one or moreembodiments of the present invention.

SUMMARY OF THE INVENTION

It is therefore a first aspect of the present invention to provide asimple and efficient novel lyophilization process for providing ateverelix-TFA lyophilizate, and wherein the use of strong organicsolvents for dissolving the teverelix-TFA is eliminated,

-   -   it is a second aspect according to the present invention to        provide a lyophilization process which does not require a filter        sterilization step for providing a sterile teverelix-TFA        lyophilizate,    -   it is a third aspect according to the present invention to        provide a teverelix-TFA lyophilizate having a higher        teverelix-TFA concentration than hitherto known,    -   it is a fourth aspect according to the present invention to        provide a teverelix-TFA lyophilizate that can be easily        resuspended in an aqueous resuspending liquid thereby providing        a homogen aqueous teverelix-TFA formulation,    -   it is a fifth aspect according to the present invention to        provide a method of adjusting the molar ratio of peptide to        counter-ion in the aqueous teverelix-TFA formulation.

These and further aspects are achieved according to the preventinvention by providing a lyophilization process for preparing ateverelix-TFA lyophilizate, said process comprises the following steps:

-   -   a) providing a lyophilization suspension by mixing teverelix and        trifluoroacetate at a molar ratio sufficient for providing an        microcrystalline teverelix-TFA suspension without formation of a        gel, and    -   b) lyophilizating the lyophilization suspension, thereby        providing a teverelix-TFA lyophilizate.

DETAILED DESCRIPTION OF THE INVENTION

A number of advantages are obtained by using the lyophilsation processaccording to the present invention. First of all, since higherconcentrations of teverelix-TFA (about 100 mg/ml), can be obtained in amicrocrystalline teverelix-TFA suspension, compared to the concentrationof teverelix-TFA dissolved in a solvent (about 10 mg/ml), asignificantly smaller lyophilization volume is required in order toobtain the same quantity of teverelix-TFA. This ensures that thelyophilization step may be conducted faster and more cost-efficient thanthe conventional lyophilization processes.

Secondly, since it is the microcrystalline teverelix-TFA suspension thatis lyophillisated, the need for using strong acids for dissolving theteverelix-TFA is eliminated. Thus, the lyophilization process accordingto the present invention provides the possibility of obtaining a “pure”teverelix-TFA lyophilizate i.e. without any undesirable residues in thecomposition, in a fast and simple manner.

In order to obtain a microcrystalline teverelix-TFA suspension, withoutformation of a gel, the inventors of the present invention have foundthat a molar ratio of teverelix to the counter-ion trifluoroacetate hasto at least 1:2.1, since a molar ratio below 1:2.1 will result information of a gel. When the molar ratio is above 1:2.2 themicrocrystalline teverelix-TFA suspension is also homogeneous.

Within the content of the present invention the term “molar ratio ofteverelix to trifluoroacetate” refers to the molar relationship betweenteverelix and trifluoroacetate, where the first number of the molarratio is the mol content of teverelix in the composition and the secondnumber refers to the mol content of TFA in the composition. Forinstance, a molar ratio of 1:2.2 means that for each mol teverelix inthe composition, said composition comprises 2.2 mol TFA, and a molarratio of at least 1:2.2 means that for each mole teverelix in thecomposition, the composition comprises at least 2.2. moltrifluoroacetate (TFA).

The obtained teverelix-TFA lyophilizate may be stored and reconstitutedwith water or another suitable aqueous solution in order to prepare anaqueous teverelix-TFA formulation that e.g. may be used as an injectablepharmaceutical formulation.

However, in order to obtain a more stable teverelix-TFA lyophilizate, orbe able to provide a specific said molar ratio in dependence of theintended use, it is desirable to lower the molar ratio, i.e. reduce thecontent of TFA in said composition, and step a) therefore comprises thefollowing additional steps:

-   -   a′) centrifuging or filtering the microcrystalline teverelix-TFA        suspension from step a), thereby providing a teverelix-TFA        pellet or a teverelix-TFA filter cake, and    -   a″) suspending said teverelix-TFA pellet or said teverelix-TFA        filter cake in an aqueous suspension solution.

Whereas the lyophilization suspension can be directly lyophilizatedwithout the centrifuging step or the filtering step, it is preferredthat it is the teverelix-TFA pellet or teverelix-TFA filter cakeobtained after the microcrystalline teverelix-TFA suspension has beencentrifuged or filtered which is suspended and then subjected to alyophilization step.

The inventors of the present invention have found that if themicrocrystalline teverelix-TFA suspension has a molar ratio above 1:2.1(which is the molar ratio in which no gel is obtained), the molar ratioin the pellet or filter cake after centrifugation will be reduced to amolar ratio between 1:1.70 and 1:1.85. For instance, the inventors foundthat when the teverelix and trifluoroacetate was mixed at a molar ratioof 1:2.2 and with a peptide concentration of 75 mg/ml, the pellet aftercentrifugation had a molar ratio of about 1:1.77.

Thus, the centrifugation/filtration and suspension steps of thelyophilization process according to the invention provides theadvantages that the resultant teverelix-TFA lyophilizate will have apredefined molar ratio which is lower than the molar ratio of themicrocrystalline teverelix-TFA suspension, thereby ensuring that themolar ratio can be adjusted to any desired molar ratio (above theinitial molar ratio) when the lyophilizate is reconstituted.Furthermore, since the lyophilizate has a reduced content of thecounter-ion trifluoroacetate, the pH value in the lyophilizate ismaintained at a value which ensures that a possible deamidation ofteverelix is reduced, whereby the stability of teverelix during storageis increased.

In order to reduce the volume on which the lyophilization is to takeplace, and at the same time ensure a high concentration of teverelix inthe resultant lyophilizate, the microcrystalline teverelix-TFAsuspension is in one embodiment centrifuged under conditions sufficientfor ensuring that a pellet is formed. The pellet is then suspended in arelatively small amount of aqueous suspension solution, and thelyophilization suspension is then the suspended pellet

However, the inventors of the present invention have found, that if ateverelix-TFA filter cake is obtained by filtering the microcrystallineteverelix-TFA suspension (instead of centrifuging said suspension andproviding a teverelix-TFA pellet), the aqueous teverelix and the excessof TFA will pass through the filter, and only the solid teverelix(microcrystals), at a low molar ratio i.e. a molar ratio between 1:1.70and 1:1.85, will be collected on the filter. Thus, use of a filteringstep for providing a teverelix-TFA cake provides a lyophilized productconsisting essentially of microcrystals from the microcrystallineteverelix-TFA suspension, and therefore provides an improved finalteverelix-TFA lyophilizate.

Any kind of filtering technique may in principal be used for obtainingthe teverelix-TFA filter cake, however it is preferred to use a pressureor vacumm filter technique using a suitable filter, e.g. a 0.45 to 0.8□m membrane filter, as such a filter is capable of both allowing theviscose teverelix-TFA suspension to pass and effectively holding themicrocrystals back.

The inventors have further found that by first providing a pellet orfilter cake and then suspending said pellet or cake in an aqueoussuspension solution, the need for using strong acids for dissolving theteverelix-TFA is surprisingly eliminated. The aqueous suspensionsolution for suspending the pellet or filter cake may in a preferredembodiment be either water or a mannitol solution, e.g. a 5% mannitolsolution, thus if small residues of the solvent remains in thelyophilisate, this will not influence the lyophilisates ability to beused in a pharmaceutical formulation.

Irrespectively of the microcrystalline teverelix-TFA suspension issubjected to a centrifugation or filtering step, it is preferably thatthe microcrystalline teverelix-TFA suspension in step a) is made ineither water, e.g. physiological water, or in a trifluroacetic acidsolution. However, if the molar ratio of teverelix to trifluoroacetateis at least 1:2.2 it is preferred that the teverelix-TFA suspension ismade in water, and if said molar ratio is below 1:2.2 it is preferredthat the suspension is made in a trifluroacetic acid solution in orderto add trifluroacetic acid to the suspension in order to obtain a molarratio of at least 1:2.2.

The molar mass of trifluoroacetate and teverelix has been calculated to:M_(TFA)=114 g/mol and M_(Tev)=1459 g/mol, and the molar ratio and/orcontent of trifluoroacetate or teverelix in the suspension canaccordingly be calculating using the following formula:

${{Molar}{ratio}} = \frac{{trifluoroacetate}{content}{in}{suspense}/M_{TFA}}{{teverelix}{content}{in}{suspension}/M_{Tev}}$

In order to ensure a high teverelix concentration in the teverelix-TFAlyophilizate, it is preferred that the concentration of teverelix-TFA inthe microcrystalline teverelix-TFA suspension in step a) is at least 100mg/ml. If the suspension thereafter is centrifugated, a high teverelixconcentration further has the advantage that less peptid will bediscarded with the supernatant.

Lyophilization is a process well known in the art, and said process willnot be discussed in details in the present application. However, inshort, the sample, i.e. the lyophilization suspension (which may eitherbe the microcrystalline teverelix-TFA suspension or the suspended pelletor the filter cake), is transferred into one or more glass container(s)and is frozen as quickly as possible, e.g. by immersing the outside ofthe container into liquid nitrogen or by using electrically poweredfreezers. Moreover, the container(s) may optionally be rotated in orderto spread and freeze the sample on a large surface area. The glasscontainer(s) with the sample is then preferably placed into an extremelylow-pressure space (vacuum) that contains a cooling coil as well. Thecooling coil acts as a condenser. The temperature of the coil is usuallylower than −50° C. Volatile compounds of the frozen sample willevaporate (sublimate) in the vacuum. The process of evaporation (in thiscase, sublimation) absorbs heat. This effect keeps the sample frozen.Evaporated molecules are captured from the gas phase by the coolingcoil, forming a frozen layer on it. At the end of the process, theteverelix-TFA remains in the container in a solid form. Since thisprocess does not cause degradation of teverelix, the lyophilizationprocess according to the invention may not only be used to concentratethe teverelix, but may also be used to preserve said peptide forlong-term storage.

A person skilled in the art will in view of the present inventionunderstand that instead of subjecting the lyophilization suspension fromstep a) to a lyophilization process, said suspension may in a modifiedprocess according to the invention be subjected to a spray-dryingprocess or similar drying process, in order to obtain a powder and/or apowder like product.

Since lyophilization is carried out on a lyophilization suspension madein water or mannitol, said suspension does not comprise any harmfulnon-volatile compounds, e.g. non-volatile acids that may can causeextreme pH-values, and salts that may result in very high ionicstrength. Accordingly, the resultant lyophilizate will not contain anyundesirable compounds, and a resuspended aqueous teverelix-TFAsuspension can safely be administrated to a patient in need of suchtreatment.

In most conventional lyophilizations the solution to be freeze-dried isinitially filtered aseptically to remove any extraneous solids andmicroorganism. However, since the viscosity of the teverelix-TFAsolution causes the sterilization filters to clog such a sterilizationstep in both laboriously and expensive. The inventors of the presentinvention have found that the teverelix-TFA lyophilizate obtained by theprocess according to the present invention may be subjected to gammasterilization, thereby eliminating the requirement for filtersterilization. Furthermore, the requirement for working under asepticconditions e.g. in a clean room, is also eliminated.

Normally a lyophilizate is stored in a substantially dry state in orderto maintain the stability of the composition. The inventors of thepresent invention have however found that if a small amount of water ispresent in the teverelix-TFA lyophilizate according to the invention,i.e. in an amount between 0.3% to 5% by weight, preferably around 1 to2% by weight, based on the total weight of the teverelix-TFAlyophilizate, an improved teverelix-TFA lyophilizate is provided whichis easier to handle, reconstitute, and accordingly use.

Without being bound by theory, the water content may provide highelectrostatic forces between particles of teverelix, which is ofimportance when the teverelix-TFA lyophilizate is handled e.g. if thelyophilizate are to be filled to a vial or a syringe chamber.

In a preferred embodiment water is present in the teverelix-TFAlyophilizate in an amount between 1% by weight and 2% by weight,preferably 1.5% by weight, based on the total weight of theteverelix-TFA lyophilizate, as this will provide a teverelix-TFAlyophilizate that retains its chemical integrity and provides a stablecomposition.

The stability provided by the invention enables a longer shelf-life atroom temperature so that the teverelix-TFA lyophilizate may be storede.g. after sterilization. The reconstitutable teverelix-TFA lyophilizatecan be packaged and stored (e.g. in a syringe or vial) for later use.

Due to electrostatic forces between dry particles of teverelix it may bedifficult to transfer the teverelix-TFA lyophilizate to a vial,container or a syringe chamber, as the particles will stick to surfacesof equipment etc. It is accordingly preferred that before the suspendedpellet or suspended filter cake is subjected to lyophilization, i.e.dried, said suspended pellet/filter case i.e. when it is in an aqueousstate is either placed in, or otherwise transferred to, a vial orcontainer in which the suspended pellet or suspended filter cake may besubjected to lyophilization. It is further preferred that said vial orcontainer also can be used for storing the resultant teverelix-TFAlyophilizate.

In a preferred embodiment the vial or container will contain a unitdosage of the teverelix-TFA lyophilizate after lyophilization. Withinthe context of the present invention the term “unit dosage” is theamount of teverelix administered to a patient in a single dosage.

The present invention also relates to a method of reconstituting theteverelix-TFA lyophilizate, and wherein said method comprises adding anaqueous reconstitution solution to the teverelix-TFA lyophilizate, andadjusting the molar ratio of the molar ratio of teverelix totrifluoroacetate by adding trifluoroacetate. Said method willeffectively and simply achieve an exact and desired molar ratio in orderto obtain the fluid, milky microcrystalline aqueous suspension ofteverelix-TFA, without formation of a gel. It is preferred that themolar ratio of teverelix to TFA after reconstitution is adjusted to atleast 1:2.1, preferably at least 1:2.2 and even more preferred at least1:2.4 by adding a sufficient amount of trifluoroacetate as this willprovide an aqueous pharmaceutical formulation that may be used directly,i.e. the formulation is ready-to-use.

The trifluoroacetate content may be added/adjusted after thelyophilizate is reconstituted, but in a preferred embodiment thetrifluoroacetate is part of the aqueous reconstitution solution, as thiswill ensure a fast and effective way of reconstituting the teverelix-TFAlyophilizate according to the invention. If desired, the aqueousreconstitution solution may contain an isotonic agent, such as mannitoland/or a pharmaceutically acceptable excipient.

The correct amount of trifluoroacetate to be added may easily becalculated for a person skilled in the art. For instance, if theteverelix-TFA lyophilizate has a molar ratio of 1:1.77, and if a molarratio of 1:2.2 is desired in the final aqueous teverelix-TFAformulation, then 0.43 mol TFA per mol teverelix present in thecomposition has to be added during the reconstitution process.

The inventors of the present invention have furthermore found that whenthe molar ratio of teverelix to trifluoroacetate is at least 1:2.1 inthe final aqueous pharmaceutical formulation, the formulation willcomprise both soluble and insoluble teverelix, thereby providing aunique bioavailablity of teverelix.

Without being bound by theory, the soluble teverelix is in the form ofan aqueous solution and in some situations, a gel. The presence of a gelwill inhibit any freely aqueous teverelix and therefore prevent, or atleast reduce, immediate release. The insoluble teverelix is in the formof microcrystals. Said microcrystals will prevent gel formation,therefore “unlocking” the aqueous teverelix. Over time the TFA in thecomposition according to the invention will be absorbed by the body,lowering the ratio, so the microcrystals subsequently turn in to gel,which forms the slow release depot. Thus, the non-gel-soluble teverelixis immediately available, providing an almost immediate onset of action,and the gel-soluble and insoluble teverelix (microcrystals) will assistin providing a sustained release of teverelix.

Accordingly, using the teverelix-TFA lyophilizate according to theinvention, it is possible to adjust the release profile of teverelixsimply by adjusting the amount of trifluoroacetate added to theteverelix-TFA lyophilizate and thereby change the ratio of insoluble tosoluble teverelix in the injected formulation.

Since teverelix is deamidated when placed in contact with acid,undesirable degradation products (impurities) will appear within thecomposition/formulation during storage. Said impurities may influencequality, safety and efficacy of the teverelix composition/formulation,thereby potentially causing serious health hazards.

The inventors of the present invention have found that the level ofimpurities are kept at an acceptable level, when the molar ratio ofteverelix to TFA is at or below 1:2.8, i.e.

when the molar content of TFA is at or below 2.8 per mol teverelix.Thus, the optimal molar ratio in the teverelix-TFA lyophilizateaccording to the invention is preferably between 1:2.2 (or 1:2.4) and1:2.8.

In a preferred embodiment the invention also relates to a kit,comprising a first package filled with a unit dosage of teverelix and asecond package filled with a reconstitution solution comprising asufficient amount of TFA for obtaining the desired molar ratio of atleast 1:2.1, preferably at least 1:2.2 and even more preferred about orabove 1:2.4, optionally with a molar ratio at or below 1:2.8 Said firstpackage may e.g. be a syringe and the second package be physicallyconnected to said syringe in order to ensure that the correct molarratio of teverelix to TFA is obtained. As one example of a first andsecond package which is physically connected to each other can bementioned a conventional dual chamber syringe for lyophilised products.Such dual chamber syringe is well known in the art.

In one embodiment said kit is arranged for providing a final fluid,milky microcrystalline aqueous teverelix-TFA suspension having a molarratio of teverelix to counter-ion of at least 1:2.1, preferably at least1:2.2 and even more preferred about or above 1:2.4, and optionally at orbelow 1:2.8. Preferably the concentration of teverelix is between 30mg/ml and about 100 mg/ml, and even more preferred between 45 mg/ml and90 mg/ml, e.g. about 75 mg/ml. The concentration of teverelix may insome situations be higher than about 100 mg/ml. The volume may bebetween 0.4 ml and 1.6 ml, e.g. about 1.2 ml. Injection givensubcutaneous and/or intramuscularly at this concentration and volume,has proven to only provide a mild injection site reaction.

The lyophilizates and formulations provided in the present invention isinexpensive to manufacture, and due to the ease of use they provide avery simple dosage regime.

EXAMPLES In order to establish the influence of the molar ratio ofteverelix to the counter-ion trifluoroacetate a number of tests wereperformed. Example 1: Preparation of Teverelix-TFA Compositions withDifferent Molar Ratio

A custom-manufactured batch of teverelix with low TFA content, Batch A,was obtained. The characteristics of the batch are shown in table 1.

TABLE 1 Purity 99.3% Teverelix content 85.56 weight-%  TFA content 10.9weight-% Acetate content  0.3 weight-% Water content  4.3 weight-%

If a composition A containing 75 mg teverelix is desired, then 88.28 mgof batch A has to be used, calculated as follows:

$\frac{75{mg}{teverelix}}{99.3/100( {\%{purity}} ) \times 85.56/100( {\%{teverelix}{content}} )} = {88.28{mg}}$

The molar ratio of teverelix to TFA in composition A can then becalculated.

The TFA content in 88.28 mg of batch A can be calculated to 88.28mg×10.9/100 (TFA content in %)=9.62 mg Since the molar mass of TFA,M_(TFA), is 114 g/mol, and the molar mass of teverelix, M_(TEV), is 1459g/mol, the molar concentration in the 75 mg teverelix composition of TFAcan be calculated to 0.084 mmol and the molar concentration of teverelixto 0.051 mmol. Thus, the molar ratio of teverelix to TFA in compositionA, is 1:1.64.

In order to prepare a number of different aqueous teverelix-TFAcompositions with different molar ratios, twenty-one samples containing44.14 mg+5% (41.93 to 46.35 mg) of composition A were accurately weighedin 2 ml glass tubes having a cap through which an aqueous solution couldbe added by means of a micropipette.

Seven TFA solutions containing TFA in 5% mannitol were prepared using aTFA composition obtained from Acros Organics, Geel, Belgium. Said TFAcomposition were 99% pure and had a density of 1.535 g/ml. Therespective solutions are shown in

Table 2.

TABLE 2 Solution A B C D E F G TFA 0 0.01 0.023 0.036 0.049 0.062 0.075mol/L

The respective aqueous teverelix-TFA compositions were prepared byadding 0.5 ml of each of the above solutions though the cap of thetwenty-one glass tubes containing 44.14 mg+5% (41.93 to 46.35 mg) ofcomposition A using a micropipette, i.e. three aqueous teverelix-TFAcompositions having the same molar ratio were prepared. The mixtureswere stirred using a vortex for 1 minute, and the solutions wereobserved visually for 10 minutes in order to establish if the desiredfluid, milky microcrystalline homogeneous aqueous suspension of theteverelix-TFA, were obtained, or if a gel was formed instead. Theresults are summarized in Table 3 below:

TABLE 3 Formation Microcrys- Formation homoge- Molar of talline of milkyneous Tubes ratio gel formation suspension suspension A1, A2, A3 1:1.64yes no no — B1, B2, B3 1:1.85 yes no no — C1, C2, C3 1:2.1  no yes yesno D1, D2, D3 1:2.36 no yes yes yes E1, E2, E3 1:2.61 no yes yes yes F1,F2, F3 1:2.86 no yes yes yes G1, G2, G3 1:3.12 no yes yes yes

The microcrystalline content of the aqueous teverelix-TFA compositionsin the No. 1 test tubes were further observed under a polarized lightmicroscope supplied by Realux, France. The results for the respectivemolar ratio are shown in FIG. 1 a -FIG. 1 g . From these observations itis clear that microcrystalline formation is not observed for the molarratios of 1:1.85 and below, thus the molar ratio of teverelix to thecounter-ion TFA has to be above at least 1:2.1 in order for the desiredmicrocrystalline formation to be initiated.

Furthermore, as is evident from table 3, a homogeneous suspension ofteverelix-TFA was not obtained until the molar ration was above 1:2.1.Thus, it is accordingly preferred that the molar ratio in both themicrocrystalline teverelix-TFA suspension used for providing thelyophilization suspension;

and the reconstituted aqueous teverelix-TFA suspension is above 1:2.1and preferably even higher such as at least 1:2.2, and even morepreferred at least 1:2.4.

Example 2: Content of Soluble Teverelix and Insoluble Teverelix inRelation to the Molar Ratio

In order to determine the content of soluble teverelix in relation toinsoluble teverelix in the respective test tubes, the No. 2 and No. 3test tubes for each molar ratio were centrifuged at 10,000 rpm for 10 to20 minutes, and the concentration of teverelix in the supernatant andpellet were measured using a HPLC analysis.

The chromatographic conditions for the HPLC analysis is shown in table4.

TABLE 4 Column Type (Aptys No) Lichrospher 100 RP18 (No128) Particlessize 5 μm Diameter 4 mm Length 125 mm Pre-Column Type Lichrocart 100RP18 Particles size 5 μm Diameter 4 mm Length 4 mm Mobile PhaseAcetonitrile/Water/TFA (35:65:0.1 V/V/V) Injector cleaningAcetonitrile/Water (50:50 V/V) Flow 1.0 mL/min Pressure Approx. 65 barsOven Temperature 30° C. Wavelength 210 nm Injection volume 10 μLInjector temperature 20° C. Retention time of Teverelix Approx. 5.6 minRun time 10 min

Two 100% standards were prepared by weighing 59.9 mg teverelix acetate(batch 080113) in a volumetric flask and completing the volume to 100 mlwith water:acetonitrile 65:35 v/v. 10 ml of this solution were completedto 50 ml with the same solvent, providing a concentration of 0.1 mg/mlteverelix peptide.

A 1% standard solution was prepared by diluting 2 ml of the 100%standard to 200 ml with the same solvent providing a concentration of0.001 mg/ml teverelix peptide.

Internal standardization was carried out using the two 100% standards.The 1% standard was used to check the linearity of the response.Recovery with the 100% standard must be in the interval 95%-105%.

The pellet obtained after centrifugation was solubilised inwater:acetonitrile 65:35 v/v, and the volume was completed to 100 mLwith the same solvent. This solution was diluted by 5 (10 mL in 50 mL)and HPLC was performed.

The supernatant was transferred to a volumetric flask and the volume wascompleted to 100 mL with the same solvent, i.e. water:acetonitrile 65:35v/v. This solution was diluted by 5 (10 mL in 50 mL) and HPLC wasperformed. The results of the HPLC analysis is shown in table 5.

TABLE 5 Supernatant - Pellet - Teverelix Teverelix Test Molarconcentration concentration tube ratio (mg/ml) (mg/ml) A2 1:1.64 52.0N/A A3 1:1.64 58.5 N/A B2 1:1.85 57.2 N/A B3 1:1.85 60.3 N/A C2 1:2.1 25.9 26.9 C3 1:2.1  26.1 25.5 D2 1:2.36 9.4 39.3 D3 1:2.36 8.3 44.9 E21:2.61 5.4 50.8 E3 1:2.61 7.2 51.6 F2 1:2.86 3.7 56.2 G3 1:2.86 3.6 58.4G2 1:3.12 1.5 53.6 G3 1:3.12 1.2 58.4

The average concentrations of each molar ratio was calculated, see table6, and the results are depicted in FIGS. 2 and 3 .

TABLE 6 Total Supernatant - Pellet - (pellet + Average Averagesupernatant) Teverelix Teverelix Teverelix Test Molar concentrationconcentration concentration tube ratio (mg/ml) (mg/ml) (mg/ml) A 1:1.6455.3 N/A 55.3 B 1:1.85 58.8 N/A 58.8 C 1:2.1  26.0 26.2 52.2 D 1:2.368.9 42.1 51.0 E 1:2.61 6.3 51.2 57.5 F 1:2.86 3.7 57.3 61.0 G 1:3.12 1.456.0 57.4

As is evident from table 5, and 6, and FIGS. 2 and 3 , the degree ofinsoluble teverelix increases when the amount of trifluoroacetateincreases in relation to teverelix, thus at a molar ratio of 1:2.1,about 50% of the pharmaceutical formulation consist of insolubleteverelix, whereas the amount of insoluble teverelix is about 82% at amolar ratio of 1:2.36 (˜1:2.4)in the pharmaceutical formulation.

Example 3: Plasma Concentration in Relation to the Molar Ratio

In order to evaluate the relevance of the molar ratio on the plasmaconcentration of teverelix, five glass vials containing different molarratios were prepared as discussed in example 1, and the test tubescomprising the aqueous teverelix-TFA compositions shown in table 7 wereprovided:

TABLE 7 Tube I II III IV V Molar 1:1.64 1:2.1 1:2.36 1:2.61 1:2.86 ratio

Five rats were tested with each molar ratio. Each rat was injected with60□1 of the respective solutions using a 25 mm 21G luer 6% regular bevelneedle (obtainable from Terumo, Leuven, Belgium) and 100□1 luer slipsyringe (obtainable from Hamilton Company, Reno, USA). Plasmaconcentrations were measured prior to administration, then at 1 h, 6 h,24 h, 48 h, 7 days, 10 days, 14 days, 21 days and 28 days followingadministration.

The peak plasma concentrations, Cmax, of teverelix after injection toeach individual rat are shown in table 8, and depicted in FIG. 4 .

TABLE 8 Test Molar Cmax Cmax tube ratio Cmax Cmax Cmax Cmax Cmax meanmedian I 1:1.64 57.6 58.8 35.4 32.5 25 41.86 35.4 II 1:2.1  96 82.6 57.450.1 n.a. 76.525 70 III 1:2.36 67.6 50 67.9 64.2 88.6 67.66 67.6 IV1:2.61 78.8 48.6 85.5 77.5 55.3 69.14 77.5 V 1:2.86 111 99.7 94.9 91.984.8 96.46 94.9

As is clear from these results the Teverelix Cmax increases until amolar ratio of 1:2.1 after which the plasma concentration issubstantially stable.

The plasma concentration over a four week period, was also measured bytaking blood samples at regular intervals.

The mean plasma levels in a four week period is shown in FIG. 5 , and itis clear that the release profile of teverelix is dependent on the molarratio. For instance a higher plasma concentration of teverelix is shownwith the suspension having a molar ratio of 1:2.1. Thus, it is possibleto adjust the release profile of teverelix simply by adjusting theamount of trifluoroacetate added to the teverelix-TFA lyophilizateduring reconstitution, thereby changing the molar ratio of teverelix totrifluoroacetate in the pharmaceutical formulation.

Clinically this offers the potential of optimizing the therapy to therequirements of individual groups of patients e.g. relating to differentindications, age and/or gender. One patient group may need an immediateonset of action, requiring a high concentration of soluble teverelix,whereas another group may require a sustained release of teverelix,requiring a low concentration of soluble teverelix. In a similar manner,different pharmaceutical formulations having different molar ratios maybe administered at different stages of a patient's treatment.Furthermore, the possibility of adjusting the molar ratio to specificneeds of different patient groups, will increase patient acceptance andcompliance of therapy.

Example 4: Stability of Teverelix in Relation to the Molar Ratio

In order to establish the influence of the molar ratio of 25 teverelixto the counter-ion trifluoroacetate on the stability of teverelix, thefollowing test was performed.

Four batches of teverelix TFA solutions were prepared with differingmolar ratios of teverelix to TFA (low: 1:1.7; mid-range: 1:2.16; high1:2.8; and extreme: 1:4.0) at two concentrations: 10 mg/mL (expressed asbase teverelix) and 1 mg/mL (expressed as base teverelix).

A reconstitutable Teverelix TFA composition, supplied as a dried powder,was obtained. The characteristics of the batch are shown in table 9:

TABLE 9 Teverelix content 79.8% TFA content 13.5% Water content 3.1%

The molar ratio of the starting material was determined using thefollowing calculation:

$ \frac{{Teverelix}{content}/{molecular}{weight}{of}{teverelix}}{{TFA}{content}/{molecular}{weight}{of}{TFA}}\Rightarrow\frac{79.8/1459}{13.1/114}  = {\frac{1}{2.16} = {1:2.16}}$

The eight batches, one for each of the four molar ratios of 10 mg/ml,and one for each of the four molar ratios of 1 mg/ml, were prepared asfollows.

Low Molar Ratio (1:1.7) at 10 mg/mL

-   -   1. 0.312 g of teverelix TFA (net weight teverelix) was        reconstituted with water for injection, making the suspension up        to 3.0 mL to form an 104 mg/mL homogenous milk suspension.        Previous investigations demonstrate that at this concentration        96% of the teverelix will form solid teverelix, therefore        approximately 300 mg of teverelix will be recovered as solid        teverelix following centrifugation.    -   2. The preparation was immediately centrifuged for 10 minutes at        10,000 rpm (8,500 g) at 4° C.    -   3. The supernatant from the centrifuged material was discarded.        Previous investigations have demonstrated that the solid        teverelix has a molar ratio of approximately 1:1.7 teverelix to        TFA.    -   4. The centrifugation pellet was resuspended with water for        injection and made up to 30 mL to form a solution of        approximately 10 mg/mL and a molar ratio of approximately 1:1.7.

Mid-Molar Range Ratio (1:2.16) at 10 mg/mL

-   -   1. 0.1 g of teverelix TFA (net weight teverelix) was        reconstituted with water for injection in a 10 mL conical flask        to make a solution of 10.0 mL volume to form a solution of        teverelix at 10 mg/mL and a molar ratio of 1:2.16 teverelix to        TFA.

High Molar Ratio (1:2.8) at 10 mg/mL

-   -   1. 0.1 g teverelix TFA (net weight teverelix) was reconstituted        with 5 mL of 0.0097 M trifluoroacetic acid in water for        injection in a 10 mL conical flask    -   2. The solution was made up to 10.0 mL with water for injection        to form a solution of teverelix at 10 mg/mL and a molar ratio of        1:2.8 teverelix to TFA.

Extreme Molar Ratio (1:4.0) at 10 mg/mL

-   -   1. 0.1 g teverelix TFA (net weight teverelix) was reconstituted        with 5 mL of 0.0252 M trifluoroacetic acid in water for        injection in a 10 mL conical flask    -   2. The solution was made up to 10.0 mL with water for injection        to form a solution of teverelix at 10 mg/mL and a molar ratio of        1:4.0 teverelix to TFA.

Low Molar Ratio (1:1.7) at 1 mg/mL

-   -   1. 0.312 g of teverelix TFA (net teverelix) was reconstituted        with water for injection, making the suspension up to 3.0 mL to        form an 104 mg/mL homogenous milk suspension.    -   2. The preparation was immediately centrifuged for 10 minutes at        10,000 rpm (8,500 g) at 4° C.    -   3. The supernatant from the centrifuged material was discarded    -   3. The centrifugation pellet was resuspended in water for        injection (final volume 300 mL) to make up a solution of        approximately 1 mg/mL and a molar ratio approximately 1:1.7        teverelix to TFA.    -   4. 10.0 mL was transferred to a 10 mL conical flask.

Mid-Molar Range Ratio (1:2.16) at 1 mg/mL

-   -   1. A 1 mg/mL solution of teverelix TFA in water for injection        was prepared

High Molar Ratio (1:2.8) at 1 mg/mL

-   -   1. 0.010 g teverelix TFA (net weight teverelix) was        reconstituted with 5 mL of a 0.001 M trifluoroacetic acid in        water for injection in a 10 mL conical flask    -   2. The volume was completed to 10 mL with WFI

Extreme Molar Ratio (1:4.0) at 1 mg/mL

-   -   1. 0.010 g teverelix TFA (net weight teverelix) was        reconstituted with 5mL of a 0.0205 M trifluoroacetic acid in        water for injection in a 10 mL conical flask    -   2. The volume was completed to 10 mL with WFI

All of the solutions were kept at lab temperature (20° C.) beforeanalyses for teverelix purity.

Samples was taken from each solution in duplicate and analysed forteverelix purity using a conventional RP-HPLC method. The chromaticconditions were as shown in table 10:

TABLE 10 Column Phenomenex Aqua C18 150 2.0 mm, 3 μm, 125 Å, LCC-012Column temperature 65° C. Autosampler temperature 4° C. Flow rate 0.3ml/min Injection volume 3 μl Run time 60 minutes Detection UV detection,226 nm

The purity of teverelix in the solutions after preparation, i.e. at timezero, is shown in table 11:

TABLE 11 10 mg/mL 1 mg/mL Molar ratio Time 0 Time: 0 1:1.7 99.47% 99.58% 1:2.16 99.45% 99.49% 1:2.8 99.48% 99.48% 1:4.0 99.47% 99.48%

In order to evaluate the stability over time, the respective solutionswere then stored in stoppered glass conical flasks in a chamber at +40°C. and a relative humidity of 75%.

After one month for the 10 mg/mL solutions, and two weeks for the 1mg/mL solutions, teverelix purity analysis was repeated using the methodalready described. The purity of the solutions after the relevantperiod, is presented in table 12 below.

TABLE 12 10 mg/mL 1 mg/mL Molar ratio Time: 1 month Time: 15 days 1:1.797.49% 98.92%  1:2.16 95.99% 98.68% 1:2.8 93.49% 98.37% 1:4.0 86.16%97.97%

The stability results are shown in FIGS. 6 and 7 , and depicts theincrease in percentage of impurities during storage according to themolar ratio of the suspension. Note that the figures show the molcontent of TFA per one mol teverelix.

From said figures it is clear that higher concentrations oftrifluoroacetate in the solutions provides significantly higherconcentrations of impurities, thus the results verify that whenteverelix is placed in contact with increasing concentrations of acid(trifluoroacetate), undesirable degradation products (impurities) willappear in small amounts and may potentially influence quality, safetyand efficacy of the formulation, thereby potentially causing serioushealth hazards. Thus, in order to obtain a stable teverelix-TFAlyophilisate, it is important to provide a composition with a lowconcentration/content of trifluoroacetate, i.e. for each mol ofteverelix the molar content of trifluoroacetate should be kept as low aspossible.

From FIGS. 6 and 7 , it can be seen that when the molar ratio ofteverelix to trifluoroacetate is below 1:2.8, (i.e. 1 mol teverelix toless than or equal to 2.8 mol TFA) in the suspension, the level ofimpurities, i.e. undesirable degradation products e.g. caused bydeamidation are kept at an acceptable level.

It is also clear from said figures, that the concentration of teverelixis also relevant for the level of impurities.

However, in order to reduce the injections volumes, it is relevant tohave suspensions comprising concentrations of teverelix of at least 10mg/ml preferably at least 30 mg/ml, thus it is not practically possiblesimply to reduce the concentration of teverelix in the final fluid,milky aqueous suspension. However, this factor makes the content of acid(trifluoroacetate) in the lyophilizate even more important duringstorage, as a low level of acid will provide a more stable product.

The compositions and formulations provided in the present invention isinexpensive to manufacture, and due to the ease of use they alsoprovides a very simple dosage regime.

Modifications and combinations of the above principles and combinationsare foreseen within the scope of the present invention.

1. A lyophilization process for preparing a teverelix-TFA lyophilizate,said process comprises the following steps: a) providing alyophilization suspension by mixing teverelix and trifluoroacetate at amolar ratio sufficient for providing a microcrystalline teverelix-TFAsuspension without formation of a gel, b) lyophilizating thelyophilization suspension from step a) thereby providing a teverelix-TFAlyophilizate, characterized in, that step a) further comprises thefollowing steps: a′) centrifuging or filtering the microcrystallineteverelix-TFA suspension thereby respectively providing a teverelix-TFApellet or a teverelix-TFA filter cake, and a″) suspending saidteverelix-TFA pellet or said teverelix-TFA filter cake in an aqueoussuspension solution.
 2. The lyophilization process according to claim 1,wherein the teverelix and trifluoroacetate in step a) are mixed at amolar ratio of at least 1:2.1, preferably at least 1:2.2.
 3. Thelyophilization process according to claim 1, wherein the pellet aftercentrifuging or filter cake after filtering has a molar ratio ofteverelix to trifluoroacetate which is lower than the molar ratio in themicrocrystalline teverelix-TFA suspension.
 4. The lyophilization processaccording to claim 1, wherein the aqueous suspension solution is wateror a mannitol solution.
 5. The lyophilization process according to claim1, wherein the microcrystalline teverelix-TFA suspension is made inwater if the molar ratio of teverelix to trifluoroacetate is at least1:2.2.
 6. The lyophilization process according to claim 1, wherein themicrocrystalline teverelix-TFA suspension is made in a trifluoroaceticacid solution if the molar ratio of teverelix to trifluoroacetate isbelow 1:2.2.
 7. The lyophilization process according to claim 1, whereinthe teverelix-TFA lyophilizate from step b) is sterilized bygamma-sterilization.
 8. The lyophilization process according to claim 1,wherein the concentration of teverelix-TFA in the microcrystallineteverelix-TFA suspension is at least 100 mg/ml.
 9. The lyophilizationprocess according to claim 1, wherein the resuspended pellet orresuspended filter cake is lyophilized or spray-dried to an extent suchthat the obtained teverelix-TFA lyophilizate comprises water in anamount of between 0.3% to 5% by weight based on the total weight of theteverelix-TFA lyophilizate .
 10. The lyophilization process according toclaim 1, wherein the lyophilization is performed on a unit dosage of theteverelix-TFA lyophilizate and/or directly in the packaging for storage.11. A teverelix-TFA lyophilizate obtainable by the lyophilizationprocess according to claim
 1. 12. A method of reconstituting theteverelix-TFA lyophilizate according to claim 11, and wherein saidmethod comprises adding an aqueous reconstitution solution to theteverelix-TFA lyophilizate.
 13. The method according to claim 12,wherein the molar ratio of teverelix to trifluoroacetate is below 1:2.1in the teverelix-TFA lyophilizate, and said method comprises adjustingthe molar ratio to at least 1:2.1 by adding trifluoroacetate.
 14. Themethod according to claim 13, wherein the molar ratio of teverelix totrifluoroacetate is adjusted to at least 1:2.2 and preferably at least1:2.4.
 15. The method according to claim 12, wherein the molar ratio ofteverelix to trifluoroacetate is adjusted to be at or less than 1:2.8.16. A fluid, milky microcrystalline aqueous teverelix-TFA suspensionobtainable by the method according to claim
 12. 17. The fluid, milkymicrocrystalline aqueous suspension according to claim 16, wherein themolar ratio of teverelix to trifluoroacetate is at least 1:2.2.
 18. Thefluid, milky microcrystalline aqueous suspension according to claim 16,wherein the molar ratio of teverelix to trifluoroacetate is at or below1:2.8.
 19. A pharmaceutical formulation comprising the fluid, milkymicrocrystalline aqueous suspension according to claim 16.